The ability to deliver nucleic acids, amino acids, small molecules, viruses, etc. (hereafter referred to collectively as “cargo”) to specific cell types is useful for various applications in oncology, developmental biology, gene therapy and in the general understanding of the mode of operation of particular proteins, nucleic acids and small molecules in a model system. There are a number of viral and nonviral delivery systems which have been developed, including vectors derived from human adenoviruses, herpes simplex viruses, adeno-associated viruses, retroviruses (Mulligan, 1993, Science 260:926-932; Berns and Giraud, 1995, Ann. N.Y. Acad. Sci. 772:95-104; Smith, 1995, Ann. Rev. Microbiol. 49:807-838) and others. Nonviral delivery systems include liposomes and conjugates of plasmid and/or DNA with agents designed to facilitate recognition of specific cell surface receptors and protect the newly introduced intracellular DNA from degradation (Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432; Curiel et al., 1991, Proc. Natl. Acad. Sci. 88:8850-8854; Wagner et al., 1992, Proc. Natl. Acad. Sci. 89:6099-6103; Zatloukal et al. 1993, Gene 135:199-207; Douglas et al., 1996, Bio/Technology 14:1574-1578; Zeigler et al., 1996, Transplantation 61:812-817; Felgner, 1997, Sci. Am. 276:102-106).
The cell recognition specificity of viruses and viral vectors is generally very high, and their ability to transfer genetic material into a target cell makes them particularly attractive candidates for the delivery of cargo to a target cell. However, there are potential risks and limitations associated with the use of viral vectors for the delivery of cargo, such as the possibility of integration into a host genome by retroviral vectors, and adverse host reactions (e.g. immunological reactions) against other viral vectors, such as adenovirus. See, e.g. Yang et al., 1995, J. Virol. 69:2004-2015.
Receptor-mediated endocytosis is widely exploited in experimental systems as a natural pathway for the targeted delivery of cargo. Endocytic pathways have been used for selective delivery of therapeutic and other biologically active agents to specific cells and to particular intracellular compartments. See generally, Shen et al., 1992, Adv. Drug. Deliv. Rev., 8:93-113; Kato and Sugiyama, 1997, Crit. Rev. Ther. Drug. Carrier Syst. 14:287-331. In theses systems, ligands to cell-specific receptors are either conjugated to cargo, for example, macromolecules (Vitetta et al., 1993, Immunol. Today 14:252-259; Kuzel and Rosen, 1994, Curr. Opin. Oncol. 6:622-626), liposomes (Kirpotin et al., 1997, Biochemistry 36:66-75; Spragg et al., 1997, Proc. Natl. Acad. Sci. USA 04:8795-8800), radioisotopes or toxins (Fitzgerald, 1996, Semin. Cancer Biol. 7:87-95) and synthetic gene complexes (Wu and Wu, 1993, Adv. Drug Deliv. Rev. 12:159-167), or expressed on the surface of viral transfection vehicles (Kozarsky and Wilson, 1993, Curr. Opin. Genet. Dev. 3:49-503; Wickham et al., Gene Ther. 2:750-756).
Early in the development of receptor-mediated delivery strategies, a ligand was used, together with a polycation (such as polylysine) for the targeting of a condensed DNA to a cell where the ligand was specific for a particular cell surface receptor. See Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432; Wu and Wu, 1988, J. Biol. Chem. 263:14621-14624; Wu and Wu, 1989, J. Biol. Chem. 264:16985-16987. These strategies suffered from the inability of the DNA to be efficiently released into the cytoplasm, although internalization was successful. The addition of endosomolytic agents, such as adenovirus, improved upon the problems associated with ligand/polycation conjugates, however simplified systems were desired. See generally, Cotton and Wagner in The Development of Human Gene Therapy 265 (Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 1999).
The identity of cellular receptors and the mode of their interaction with a ligand-presenting vehicle determine the cell specificity of the delivery system and the intracellular localization of the transported molecules. See Shen et al., 1992, Adv. Drug. Deliv. Rev. 8:93-113 and Basu, 1990, Biochem Pharmacol. 40:1941-1946. This information is useful in the development of simplified methods for delivery. However, these methods are limited by the ability to transfer sufficient quantities of the molecules to specific cells in vivo, although they have proven effective in vitro. Sato et al., 1996, Adv. Drug. Deliv. Rev. 19:445-467. The application of these methods in vivo are limited by several factors, principally the low targeting efficiency of receptor-mediated delivery systems.
Another simplified synthetic system utilized short synthetic peptides based on the sequence thought to be important for membrane fusion by influenza hemagglutinin (Wagner et al., 1992. Proc. Natl. Acad. Sci. 89:7934-7938). The inclusion of these peptides into condensed-DNA complexes allowed for improved simplified delivery of the DNA to a cell. However, the limitation of this method was the affinity of the peptide for numerous cell types which also may translate into an inability to transfer sufficient quantities to a specific target cell.
One approach to improving the ability to transfer sufficient quantities of cargo to specific cells is to identify novel cell-targeting ligands, which increase the rate and specificity for the transport of molecules. The first protein discovered having such transduction properties was the HIV transactivator protein, TAT. See Green & Lowenstein, Cell, 55:1179-1188 (1988); Frankel & Paho, (Cell 55:1189-1193 (1988). Subsequently, an 11 amino acid transduction domain in TAT (TAT-PTD) responsible for the observed transduction properties was identified, based on its high basic residue content. See Fawell et al., Proc. Natl. Acad. Sci. USA 91:664-668 (1994). It has been shown that fusion protein constructs containing TAT-PTD are capable of delivering proteins to a wide spectrum of cell types both in vitro and in vivo. See Nagahara et al., Nat. Med. 4:1449-52 (1998); Vives et al., J. Biol. Chem. 272:16010-17 (1997) Shwarze et al., Science 285:1569-72 (1999); Vocero-Akbani et al., Nat. Med. 5:29-33 (1999); Moy et al., Mol. Biotechnol. 6:105-13 (1996). It is not known however if TAT-PTD will be effective in all cells and with all fusion constructs. It is possible that TAT-PTD will elicit an immune response in subjects to which it is administered. See Schwarze & Dowdy, TiPS 21:45-48. Furthermore, the half-life of TAT-PTD may vary in different cells and subjects which could also adersely-effect its transduction efficiency. See Schwarze & Dowdy, TiPS 21:45-48.
In addition, a class of peptides, called penetratins, which have translocating properties and are capable of carrying hydrophilic compounds across the plasma membrane have recently been identified. For example, Derossi et al., 1998, Trends in Cell Biology 8:84-87, have isolated a 16 residue peptide (called penetratin-1, Ant PTD, or AntP) possessing translocation properties, corresponding to amino acids 43-58 of the homeodomain of ANTENNAPEDIA, a Drosophila transcription factor which is internalized by cells in culture. The 16 residue peptide has translocation properties equivalent to those of the full length homeodomain. Derossi et al. have shown the ability of the 16 residue peptide to intracellularly deliver oligonucleotides and oligopeptides attached thereto. However, this method is limited in that oligonucleotides greater than 55 bases long and oligopeptides greater than 100 amino acids long were not shown to be efficiently delivered. Additionally, the peptide-oligonucleotide and peptide-oligopeptide hybrids may be insoluble. Furthermore, delivery was inhibited by the release by cells (particularly dying cells) of DNA into the extracellular matrix which binds to the peptide and inhibits internalization. These peptides are also susceptible to the problems of specificity and affinity for particular cell types.
Similarly, Villaverde et al. have isolated a short peptide which contains the cell attachment motif of foot and mouth disease virus (FMDV). Villaverde et al., 1998, Biotechnology and Bioengineering 59:294-301. This peptide targets a specific receptor and the mechanism of import is also receptor mediated. Villaverde et al. demonstrated that the attachment of the FMDV peptide to β-galactosidase (βgal) facilitated the uptake of βgal into cells in vitro. The attachment of the peptide was either at the n-terminus of βgal or at an internal loop of βgal. Internal attachment provided superior internalization of βgal, and attachment of multiple copies further increased the amount of internalization. This peptide demonstrated varying affinity for different cell lines and therefore is likely to work efficiently with only particular target cells.
Elliot & O'Hare (Cell 188:223-233 (1997)) have shown that VP-22, a 38 kDa tegument protein from herpes simplex virus type 1 (HSV-1) also possesses the ability to transduce attached molecules across cell membranes and that residues 267-300 of VP-22 are required, but may not be sufficient, for transduction. Since the region responsible for transduction has not yet been identified, current approaches using VP-22 have been directed to fusing the entire VP-22 protein to a molecule to facilitate the transduction of that molecule. This has several disadvantages including a greater likelihood that the fusion protein (1) will be more readily degraded in cells, (2) will be harder to produce due to solubility problems, and (3) will elicit an immune response in a subject. In addition, there is little data about the efficiency of transduction using VP-22 linked to another molecule. See Schwarze & Dowdy, TiPS 21:45-48.
Therefore, there is a need for a simplified, improved delivery means for delivering cargo, such as polypeptides, polynucleotides, small molecules, plasmids and viruses to cells which demonstrates high efficiency transfer of the cargo to a wide variety of cell types. There is also a need for a method for isolating such improved means (e.g. peptides) for the delivery of cargo into a wide variety of cell types at high efficiency.